Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2012, Article ID 701261, 6 pagesdoi:10.1155/2012/701261

Research Article

In Vitro Screening of Medicinal PlantsUsed in Mexico as Antidiabetics with Glucosidase andLipase Inhibitory Activities

Guillermo Ramırez,1, 2 Miguel Zavala,3 Julia Perez,3 and Alejandro Zamilpa2

1 Biological and Health Sciences Ph.D. Program, Division of Biological Sciences and Health, Universidad AutonomaMetropolitana-Xochimilco, 14387 Mexico, DF, Mexico

2 Southern Biomedical Research Center (IMSS), Argentina 1, Col. Centro, 62790 Xochitepec, MOR, Mexico3 Department of Biological Systems, UAM-Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, 04960 Mexico, DF, Mexico

Correspondence should be addressed to Alejandro Zamilpa, azamilpa 2000@yahoo.com.mx

Received 27 May 2012; Revised 20 August 2012; Accepted 27 August 2012

Academic Editor: Benny Tan Kwong Huat

Copyright © 2012 Guillermo Ramırez et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

This work shows the inhibitory effect on glucosidase and lipase enzymes of 23 medicinal plants described as traditional treatmentsfor diabetes in several Mexican sources. Hydroalcoholic extracts of selected plants were evaluated at 1 mg/mL for glucosidase and0.25 mg/mL for lipase inhibitory activities, respectively. Camellia sinensis, acarbose, and orlistat were used as positive controls.Dose-response curves were done with the most active species. Sixty percent of all tested extracts inhibited more than 25% ofα-glucosidase activity. C. sinensis displayed an inhibition of 85% (IC50 = 299 μg/mL), while Ludwigia octovalvis and Iostephaneheterophylla showed the highest inhibition (82.7 %, IC50 = 202 μg/mL and 60.6%, CI50 = 509 μg/mL, resp.). With respect to lipaseactivity, L. octovalvis and Tecoma stans were the most inhibiting treatments (31.4%, IC50 = 288 μg/mL; 27.2%, IC50 = 320 μg/mL),while C. sinensis displayed 45% inhibition (IC50 = 310 μg/mL). These results indicate that a high proportion of plants used inMexico as treatment for diabetes displays significant inhibition of these digestive enzymes.

1. Introduction

Since the second half of the 20th century, most societiesare experiencing an epidemic-like increase in chronic degen-erative diseases, mainly cardiovascular, cancer, and type 2diabetes mellitus. In 2008, type 2 diabetes mellitus (t2DM)world cases were estimated at 348 million individuals, witha striking increase among children and adolescents living inlow- and middle-income countries [1]. Such global surveysshow that in spite of many important discoveries aboutthe pathophysiology of the disease, reported in the last 15years, its incidence continues to climb and surpasses even theboldest projections.

Several new types of drugs have been introducedas treatments in the last decade (dipeptidyl peptidase-4inhibitors, glucagon-like peptide-1 analogs, and cannabinoidreceptor type 1 antagonists) and it seems that glucoserenal reabsorption inhibitors will be the next available

pharmacological therapy [2]. While bariatric surgery isthe most successful approach in some cases, this measureis invasive, somewhat risky (0.5–3% mortality), and veryexpensive [3]. Glucosidase and lipase inhibitors have beenavailable for a long time as prescription medicines, but theiruse is infrequent as a treatment for diabetes. The leadingglucosidase inhibitor, acarbose, is used scarcely due to itslow efficacy in decreasing glycemic levels; the lipase inhibitororlistat is approved for weight loss but is not indicated as adiabetes control measure. Both compounds elicit unpleasantside effects and are not well accepted by both patientsand physicians [4]. On the other hand, it has been shownthat some plant preparations, containing glucosidase and/orlipase inhibitors, are devoid of these side effects but are stillclinically useful (touchi, green tea) [5, 6]. Our interest inglucosidase inhibitors led us to search for lipase-inhibitingspecies, following the glucolipotoxicity hypothesis, whichstates that chronic or postprandial glucose and free fatty

2 Evidence-Based Complementary and Alternative Medicine

acids, in supraphysiological blood concentrations, contributeto beta cell failure [7]. This work explores whether a sampleof medicinal plant extracts, selected by the Mexican eth-nomedical knowledge as “antidiabetic,” contain significantlevels of both inhibiting enzymatic activities and if these invitro assays are able to identify novel candidates for furtherphytochemical and in vivo pharmacological analyses aimedat developing prototypes of phytodrugs for diabetes control.

2. Materials and Methods

2.1. General. Corn starch (S-4186); 5,5′-dithiobis(2-nitrob-enzoic acid) (DTNB, Ellman’s reagent; D-8130); pan-creatic lipase (type II, crude, from porcine pancreas,L-3126); 2,3-dimercapto-1-propanol tributyrate (DMPTB,97%; cat : 282413); albumin (fatty acid free, bovine, A-6003) were obtained from Sigma-Aldrich (St. Louis, MO,USA). Acarbose (Sincrosa, Alpharma, Mexico City, Mexico)and orlistat (Lysthin, PsicoFarma, Mexico City, Mexico)were used as positive controls. Glucose was measured witha quantitation kit (Glucosa-TR) from Spinreact (Girona,Spain). Solvents were obtained from Merck (Darmstadt,Germany); miscellaneous chemicals (salts, buffers, solvents)were purchased either from Merck or Sigma.

2.2. Bibliographical Sources Used for Plant Selection. Theethnomedical compilations of Cano [8], Aguilar and Xolalpa[9], Andrade-Cetto and Heinrich [10], and Romero-Cerecero et al. [11] were consulted to select species usedas treatment for diabetes in Mexico. L. octovalvis wasincorporated in view of information from healers and plantdealers from the south of the state of Morelos, Mexico(Xochitepec, Jojutla, and Zacatepec areas).

Most of the 23 vegetal species were collected in the state ofMorelos, Mexico, and voucher specimens were deposited atthe IMSS Herbarium (IMSSH), where identity was assessedby Professor Abigail Aguilar, curator. Annona squamosa L.was identified in situ by Juan Carlos Juarez Delgado fromHUMO Herbarium, Autonomous University of Morelos(UAEM). Camellia sinensis (Yamamotoyama, Pomona, USA)was bought at a Japanese specialty store and used as a vegetalpositive control (CsHAE).

2.3. Preparation of Hydroalcoholic Extracts. Vegetal materials(aerial parts, leaves, or seeds) were dried in a dark roomat 25–30◦C for 15 days. The dry material was ground toobtain 4–6 mm particles (diameter). Samples of 25 g of allthese plants (20 g for Hintonia latiflora) were exhaustivelyextracted by maceration with 250 mL of an ethanol solution(60%) at 50◦C for 2 hours (three times). After filtration,the solvent was removed under reduced pressure distillation.Semisolid extracts were finally lyophilized and stored at4◦C in air-tight centrifuge tubes until needed. Most speciesproduced yield over 20% (Table 1).

2.4. Glucosidase Inhibition. Glucosidase inhibition assayswere performed in quadruplicate as previously reported[12], at an extract concentration of 1 mg/mL. In brief,

cornstarch (12.5 mg/mL) was digested by crude enzyme(homogenate from Sprague Dawley rats’ intestinal mucosa)at 37◦C for 10 minutes and released glucose was quantifiedby a glucose oxidase-based clinical reagent (SPINREACT),following manufacturer’s directions. All inhibitions werecalculated as percentage of uninhibited control reactions.

2.5. Lipase Inhibition. Lipase inhibition assay was adaptedfrom the method reported by Choi et al. [13]. The assayis based on the spectrophotometric quantification of freethiols with chromogenic 5, 5′-dithiobis(2-nitrobenzoic acid)(DTNB, Ellman’s reagent), released by porcine pancreaticlipase from the 2,3-dimercapto-1-propanol tributyrate sub-strate (DMPTB, 97%).

The reaction mixture contained 0.2 mM DMPTB in50 mM TRIS-HCl, pH 7.2, 2 mM CaCl2, 0.1 M NaCl, 0.06%Triton X-100, and 0.8 mM DTNB (in DMSO).

The porcine lipase was prepared as a stock at 10 mg/mLin TRIS-HCl 20 mM, pH 6.2, bovine serum albumin (BSA;1 mg/mL) and 0.1 M NaCl, and stored at −80◦C. Theworking samples of enzyme were diluted to 2 mg/mL in BSA(1 mg/mL), kept at 4◦C, and were used during 4-5 hours. Theassays were performed at 37◦C and were started by adding 10microliters of enzyme solution to 790 microliters of reactionmix, containing 0.25 mg/mL of plant extract in 50% DMSOor controls. The absorbance changes were recorded for up to6 minutes at 412 nm, plotted in Excel (Microsoft), and theinitial slope was employed as the velocity of the reaction.

2.6. HPLC Analysis. Solutions (3 mg/mL) of the most activeextracts were analyzed using a HPLC system (Waters Co.,Milford, MA, USA) with a photodiode array detector (Waters2996). Separation was carried out using a RP-18 Supersphere(Merck) column (120 mm× 4 mm; 5 μm) with the followingsolvent ratios for the mobile phase, where solvent A iswater and solvent B corresponds to acetonitrile: A : B =100 : 0 (0–3 min); 90 : 10 (4-5 min); 80 : 20 (6–9); 0 : 100 (10–13 min); 100 : 0 (14-15 min). The flow rate was 1 mL/min anddetection wavelength was scanned at 190–600 nm. The majorcompounds were analyzed according to their UV spectra andretention time.

3. Results

The Ethnobotanical Veracruz Atlas [8] listed 49 species as“antidiabetic” while Aguilar and Xolalpa [9] and Andrade-Cetto and Heinrich [10] reported 178 and 306 species,respectively. Romero-Cerecero [11] reports 64 species usedin the state of Morelos. Ludwigia octovalvis (“clavillo”) wasfound to be mentioned as a species of emerging local use bysome diabetic patients and plant dealers. Species mentionedin at least two sources were selected, acquired, and processedto obtain their hydroalcoholic extract.

The test concentration of the extracts was adjusted toobtain a 0–85% range of glucosidase inhibition. As shown inTable 1, the vegetal positive control using C. sinensis hydroal-coholic extract (CsHAE) inhibited α-glucosidase activity

Evidence-Based Complementary and Alternative Medicine 3

Table 1: Alpha-glucosidase and lipase inhibition of 23 medicinal plants used in Mexico for diabetes trearment.

Scientific name Local name Yield % glucosidase % inhibitiona Lipase % inhibitionb

Acacia farnesiana (L.) Willd. Huizache 19.0 21.0± 3.1 26.6± 1.76

Achillea millefolium L. Milenrama 27.3 52.3± 4.1 11.5± 2.67

Annona squamosa L. Anona 16.4 22.3± 1.6 14.4± 3.0

Artemisia absinthium L. Ajenjo 15.5 67.7± 3.7 25.2± 2.14

Bidens pilosa L. Aceitilla 16.2 41.8± 1.7 13.6± 3.61

Calea ternifolia Kunth Prodigiosa 18.4 61.1± 1.7 11.8± 0.18

Camellia sinensis (L.) Kuntze Te Verde 35.5 85.0± 1.3 45.6± 4.31

Cecropia obtusifolia Bertol. Guarumbo 18.6 28± 1.9 19.9± 5.4

Hintonia latiflora (Sesse & Moc. ex DC.) Bullock Copalchi 41.3 39.2± 3.5 14.4± 2.8

Crataegus mexicana Moc. & Sesse ex DC. Tejocote 27.5 38.6± 1.6 ppt

Eucalyptus globulus Labill. Eucalipto 22.5 33.6± 3.0 11.3± 0.4

Guazuma ulmifolia Lam. Guacima 15.4 23.0± 3.9 13.1± 1.37

Iostephane heterophylla (Cav.) Benth. Zacapal 24.3 60.6± 1.5 15.1± 1.91

Justicia spicigera Schltdl. Muicle 22.1 0.4± 1.5 12.7± 4.3

Lepidium virginicum L. Lentejita 17.4 18.0± 1.1 9.6± 1.2

Ludwigia octovalvis (Jacq.) P.H.Raven Clavillo 37.0 82.7± 1.9 31.4 ± 4.31

Marrubium vulgare L. Marrubio 19.4 31.1± 2.3 1.8± 2.5

Persea americana Mill. Aguacate 27.8 17.9± 2.9 6.3 ± 1.5

Piper sanctum Miq. Hoja santa 22.2 10.5± 0.8 15.1±2.2

Psidium guajava L. Guayaba 24.5 39.5± 3.0 ppt

Ricinus communis L. Higuerilla 24.3 58.0± 1.9 14.4± 2.1

Tamarindus indica L. Tamarindo 18.8 30.1± 2.2 ppt

Taraxacum officinale Webb Diente de Leon 22.1 12.0± 1.5 5.0± 1.3

Tecoma stans (L.) Juss. ex Kunth Tronadora 25.6 32.3± 1.7 27.2± 5.3aPercentage of α-glucosidase inhibition was calculated at t = 10 min, whereby the reaction = (mean free glucose in sample/mean free glucose in uninhibited

control) × 100.bPercentage of lipase inhibition was calculated using the slope at t = 2–5 min, whereby the reaction = (mean slope in sample/mean slope in uninhibited control)× 100.ppt: sample precipitation strongly interfered with lipase assay.

significantly (IC50 = 299μg/mL). Acarbose displayed >95%inhibition at 10 μg/mL.

We chose an initial concentration of 1 mg/mL−1 forthe screening stage. The most active species (inhibition >50%, Table 1) were further tested at a lower concentration(0.5 mg/mL−1, Table 2) and the two species with the highestinhibitory activity (L. octovalvis and Iostephane heterophyllaBenth) were compared with the control (Camellia sinensis)in a dose-response curve (Figure 1). L. octovalvis showedthe lowest IC50 (202 μg mL−1), followed by C. sinensis(299 μg mL−1) and I. heterophylla (509 μg mL−1), respectively(Table 2).

Then we examined the effects of all selected extractson lipase inhibition activity evaluated at 0.25 mg/mL. Inthis case, the pharmaceutical drug orlistat and CsHAE wereused as reference. An initial concentration of 0.25 mg/mLwas chosen for all plant-screening purposes, since higherconcentrations of some extracts strongly absorbed at theemployed wavelength. Under these conditions, the referenceextract (C. sinensis) displayed 45.6% inhibition of thisdigestive enzyme. The most active species were comparedwith the control (Camellia sinensis) in a dose-response curve(Figure 2). C. sinensis, L. octovalvis, and Tecoma stans (L.)

0102030405060708090

100

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Figure 1: Dose-response curves of the hydroalcoholic extracts fromCamellia sinensis (©), Ludwigia octovalvis (�), and Iostephaneheterophylla (�) leaves in the in vitro α-glucosidase inhibitionmodel. x-axis = log concentration in μg·mL−1; y-axis = inhibitionpercentage.

H.B. & K. gave similar IC50 values (310, 288, and 320 μg/mL).In these conditions, orlistat gave an IC50 = 142 ng/mL.

The extracts of the reference plant and the most activespecies: Camellia sinensis, Ludwigia octovalvis, Iostephaneheterophylla, Acacia. farnesiana, Artemisia absinthium, and

4 Evidence-Based Complementary and Alternative Medicine

Table 2: α-glucosidase the 10 most active medicinal plants used in Mexico for diabetes treatment.

Scientific name Local name α-glucosidase % inhibition (0.5 mg mL−1) IC50 (μg mL−1)

Artemisia absinthium Ajenjo 45.0 ± 1.7 n.d.

Achillea millefolium Milenrama 22.9 ± 1.7 n.d.

Calea ternifolia Prodigiosa 39.8 ± 2.1 n.d.

Camellia sinensis∗ Te Verde 67.3± 0.8 299

Iostephane heterophylla Zacapal 51.4 ± 2.5 509

Ludwigia octovalvis Clavillo 61.3±1.4 202

Ricinus communis Higuerilla 43.4 ± 2.4 n.d.

n.d.: no determined, ∗reference plant drug control. Percentage of α-glucosidase inhibition was calculated at t = 10 min as 100% reaction, whereby the reaction= (mean free glucose in sample/mean free glucose in control) × 100.

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Figure 2: Dose-response curves of the hydroalcoholic extracts fromCamellia sinensis (©), Ludwigia octovalvis (�), and Tecoma stans(�) leaves in the in vitro lipase inhibition model. x-axis = logconcentration in μg·mL−1; y-axis= inhibition percentage.

Tecoma stans were analyzed by DAD-HPLC. The obtainedspectra were interpreted by the presence of λ max 220, 255,275, 325, 340, and/or 360 nm. The profile of C. sinensis showsλ max = 220 and 275 nm, associated with catechins, com-pounds widely reported in this species [14]. Interestingly, L.octovalvis has peaks with similar UV absorption spectrum(λ max = 220 and 275 nm), and also others characteristicof flavonols (λ max = 210, 255, and 355 nm; Figure 3). Bothspecies show very high inhibition activities. The A. farnesianaand T. stans UV spectrum profiles show flavonol and flavonepatterns (λ max = 210, 255, 350 nm and 210, 275, 330 nmresp.), while A. absinthium, C. ternifolia, and I. heterophyllahavea profile with peaks associated with caffeoyl derivatives(λmax = 210 and 325 nm) [15, 16].

4. Discussion

The scientific literature contains many reports of the invivo antidiabetic activities (mostly hypoglycemic or anti-hyperglycemic) of medicinal plants. Nevertheless, only fewof them describe some action mechanism present in theextract. This survey was designed to search for the inhibitionof two enzyme activities in a sample of ethnomedicallyselected species, considering that they may be part of theirantidiabetic properties. This approach is aimed at mildlyreducing carbohydrate and lipid digestion and absorption,

thus decreasing the hyperglycemia and hyperlipemia peaks.Both measures have been shown to be useful in decreasingthe risk of developing diabetes [17, 18].

Besides previously reported species, we obtained infor-mation from sources in the southern area of the state ofMorelos, on the use of Ludwigia octovalvis as an antidiabetic.This phenomenon may reflect the empirical search of thepopulation to find additional therapeutic resources fordiabetes control.

Two species were found with the highest levels of inhibi-tion in both activities: Artemisia absinthium (ajenjo) and L.octovalvis (clavillo). The first plant has many traditional uses:It is reported as an antiparasitic, antimicrobial, and hepato-and neuroprotective. While this plant has been reported asrich in flavonols (kaempferol and quercetagetin derivatives)and essential oils like thujone, in this work we mainly founda high level of caffeoyl derivatives [19].

L. octovalvis (Jussiaea suffruticosa) was reported byMurugesan et al. [20] as having antidiabetic activities innormal and alloxanized rats, but no action mechanism wasproposed and no further research has been published to ourknowledge. In the state of Morelos, it is employed as aninfusion for dysuria due to prostate hyperplasia; this use isshared by some regions of the neighboring state of Guerrero.This plant contains C-glycosylated flavones like orientin,isoorientin, vitexin, and isovitexin, mainly [21]. Accordingto our results, we found this kind of flavonoids in thehydroalcoholic extract, together with a high concentration ofcompounds with λ = 220, 275 nm. They could correspondto catechins [14].

Concerning the lipase inhibiting activity, three speciesyielded similar high values: Artemisia absinthium, Acaciafarnesiana (L.) Willd, and Tecoma stans (25.2, 26.6, and27.2% inhibition, resp.). Recently, Ikarashi et al. reportedthe presence of both inhibitory activities in the bark ofAcacia mearnsii [22], identifying a catechin-rich preparationas the main active fraction. We found that leaves of A.farnesiana are also very active in lipase inhibition but lowin glucosidase inhibition activities. In this case, the activeextract mainly contains flavonol and flavone-like peaks(λ max = 210, 255, 355; 216, 271, 336 nm) [23]. Tecomastans has been employed and studied as an antidiabetic fordecades; we described the presence of glucosidase activity[12] and now we report the presence of a strong lipase

Evidence-Based Complementary and Alternative Medicine 5

300 (nm)

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Figure 3: HPLC chromatograms of the reference plant and the most active species—(a) C. sinensis, (b) L. octovalvis, (c) I. heterophylla, (d)A. farnesiana, (e) A. absinthium, and (f) T. stans.

inhibiting activity. Chromatographic analysis of this plantindicated a major concentration of caffeoyl derivatives andflavone-like compounds.

The in vitro assay employed allowed us to identify highlyactive species in a fast, economical, and sensitive way. Thisapproach does not substitute in vivo testing but acts as aprefilter of the chosen enzymatic activity and directs theanimal models that, otherwise, may render negative results[24]. The lipase assay employed is sensitive and is not affectedby zwitterionic compounds present in the extracts, but maybe interfered by colored substances and free thiols, limitingits usefulness in some botanical species.

In conclusion these results support the ethnomedical useof some plants reported by the Mexican traditional medicine

and yield information about one of their action mechanismsthat could be of immediate use to traditional healers.Although the major compounds in the most active speciescorrespond to catechins, flavonols, flavones, and caffeoylderivatives, these antidiabetic plants are being subjectedto a detailed phytochemical and pharmacological study toidentify their active compounds.

Acknowledgments

The authors are indebted to Abigail Aguilar Contreras,M.S., Director of IMSSM Herbarium, and to Juan CarlosJuarez Delgado, from the HUMO Herbarium (University ofMorelos), for their support in identifying the plant species.

6 Evidence-Based Complementary and Alternative Medicine

This paper is taken in part from the Ph.D. thesis of GuillermoRamırez.

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MEDIATORSINFLAMMATION

of

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Behavioural Neurology

EndocrinologyInternational Journal of

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Disease Markers

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BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Immunology ResearchHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Research and TreatmentAIDS

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Gastroenterology Research and Practice

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Parkinson’s Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttp://www.hindawi.com